Area of Science: 02 Wnt is a morphogen released from signaling cells to transduce signals to other cells over large distances. Wnt signaling is extensively involved in developmental processes, including cell polarity, fate specification, and organogenesis, and is conserved from Caenorhabditis elegans to humans. Aberrant Wnt signaling has been implicated in many types of cancers. Wnt proteins and receptors are promiscuous in binding, and each of the coexisting multiple Wnt pathways elicits distinct cell responses. Conventional methods are ineffective in studying Wnt signaling due to the functional redundancy of Wnt signaling members as well as the difficulty in working with the small and heavily modified Wnt protein in vitro. Despite intense efforts, the mechanisms of Wnt secretion, gradient formation, and Wnt signal specificity are poorly understood. We propose to genetically encode unnatural amino acids (UAAs) into proteins in C. elegans, and to tailor UAAs with novel chemical and physical properties for new and precise studies of the Wnt signaling pathway directly in vivo. Fluorescent UAAs will be encoded to image the localization and trafficking of Wnt proteins, which cannot be tagged by bulky fluorescent proteins. Photocrosslinking UAAs will be used to covalently lock interacting Wnt molecules in vivo with high sensitivity and specificity for identification. These strategies will enable us to visualize Wnt export and trafficking in vivo, to untangle the multiple Wnt pathways, and to identify novel ligands or receptors. This study represents the first attempt to genetically encode UAAs in a multicellular organism. The success of this approach will provide a new set of methodologies with the potential to revolutionize the investigation of various biological and biomedical problems directly in vivo. New insights into the functions of Wnt secretion and receptor activation may help identify and ameliorate Wnt-related pathologies as well as implicate this machinery in other known diseases.